Home Protocols Open Chromatin Labeling and NicE-seq library construction using Nt.CviPII

Open Chromatin Labeling and NicE-seq library construction using Nt.CviPII

Open Chromatin Labeling

Harvest cells and count to determine the cell density using automated cell counter or hemocytometer.
Dilute the sample to obtain the desired number of cells in the tube.
Crosslink the cells using 1% formaldehyde for 10 min at room temperature, followed by quenching the fixation using 125 mM Glycine.
Wash the cells using 1X PBS to remove residual formaldehyde.
Isolate nuclei by resuspending cells in cytosol extraction buffer (15 mM Tris-HCl pH 7.5, 5 mM MgCl2, 60 mM KCl, 0.5 mM DTT, 15 mM NaCl, 300 mM sucrose, and 1% NP40) and incubating on ice for 10 min with occasional agitation.
Precipitate the nuclei by spinning at 1000 X g, 4˚C for 5 minutes and discard the supernatant.
Label open chromatin by incubating the nuclei in 2.5 U of Nt.CviPII (NEB #R0626S), 10 U of DNA Polymerase I (NEB #M0209S) and 30 μM of each dNTP including 6 μM of biotin-14-dATP (Invitrogen, 19524-016) and 6 μM of biotin-16-dCTP (ChemCyte, CC-6003-1) in 200 μl of 1X NEBuffer 2 at 37˚C for 2 hours.
Stop the labeling reaction by adding 20 µl of 0.5 M EDTA and 2 µg of RNase A to the reaction and incubate for 30 min at 37˚C.
De-crosslink the DNA-protein crosslinking by adding 20 µl of Proteinase K (NEB #P8107S) and 20 µl of 20% SDS to the reaction and incubating overnight at 65˚C.
Extract biotin labeled genomic DNA using phenol chloroform method.

Fragment the biotin labeled genomic DNA into 150 bp fragments using Covaris^®.
Generate libraries with fragmented DNA (up to 1 µg) using the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645S).
Following adaptor ligation, without further purification, add 50 µl of streptavidin magnetic beads (blocked with 0.1% cold fish gelatin at 4˚C) to the library, resuspended in 1 ml of B&W buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 2 M NaCl) and incubate at 4˚C for 2 hours with end-over-end mixing.
Wash the streptavidin bead bound library four times on the magnet using B&W buffer supplemented with 0.05% Triton X-100 and once with 1X TE plus 0.05% Triton X-100.
Finally, resuspend the beads in 40 µl of nuclease-free water and use 4 µl for library amplification as per the guidelines given in NEBNext Ultra II DNA Library Prep Kit.